To develop properly, organisms must not only establish defined patterns of gene expression that specify tissue identity, but also must maintain those gene expression patterns. It is known that one set of mechanisms establishes appropriate expression patterns of master regulatory factors, such as Hox genes, while a second set of mechanisms maintains those expression patterns from early in embryonic development until death of the organism. One family of genes that is required for maintenance, and that functions primarily to maintain genes in a repressed state, is the Polycomb-Group (PcG) of genes. There are at least two major regulatory complexes that contain PcG genes and that are conserved in function and in core composition from Drosophila to humans. The long terms goal of the studies proposed here is to understand the function of PcG complexes (termed 'PRCV and 'PRC2'), and to understand the mechanisms that target these complexes to specific genes and cause these complexes to repress expression in a mitotically heritable manner. We propose to functionally characterize PRC complexes, with a focus on understanding how mammalian complexes work on mammalian targets such as the HOX loci. Drosophila is used as a model system for initial characterization because of the wealth of data on PcG function in flies. The Aims are: 1) Biochemical characterization of PRC1 and PRC2 complexes, with a goal of understanding the repressed state and the mechanisms that might target that state; 2) Characterization of a novel class of small RNAs and their potential role in specifying PcG function; 3) Characterization of the chromatin structure of mammalian PcG targets in cultured cells using a recently developed long-range chromatin mapping technology. [unreadable] [unreadable] [unreadable]